Various fluorescence applications are known such as fluorescence anisotropy imaging microscopy (FAIM), and fluorescence lifetime imaging microscopy (FLIM).
Fluorescence anisotropy imaging microscopy (FAIM) concerns the study of molecular orientation and mobility using linearly polarized light. The linearly polarized light preferentially excites fluorophores which have a dipole orientation (absorption transition moment (ATM)) similar to the plane of the electric-field of the linearly polarized light. The fluorophores which have a dipole orientation orthogonal to the plane of the electric-field are not excited, this is known as photo selection. As the fluorophores rotate due to their environment and undergo other processes, the spontaneously emitted light becomes more depolarized. The degree of depolarization that has occurred can be measured by separating the emitted light into orthogonal linear components. The two main contributing factors to the depolarization are rotational diffusion and Førster resonance energy transfer (FRET). This is a particularly useful technique as FAIM can therefore provide spatially resolved information on rotational mobility, molecular binding, or clustering of fluorescently labeled molecules, without dependence on signal intensity.
Anisotropy can be measured using both steady-state FAIM and/or time-resolved FAIM.
With steady-state FAIM, the degree of anisotropy is measured by taking an average of the polarized fluorescence during the excitation time (i.e. exposure time). The steady state FAIM does not allow any information into how the degree of anisotropy changes with respect to time during the excitation (i.e., exposure time). Steady state FAIM is useful to compare cellular systems, as cells with a high degree of proximity will present a lower average degree of anisotropy than cells which are further apart.
Time-resolved FAIM, allows the change in the degree of anisotropy to be measured with respect to time. Upon the excitation of a fluorophore tagged to a cell oriented in the same direction as the incident polarized light, the fluorophore will fluoresce, wherein the fluorescence has a high degree of polarization (i.e. indicating high anisotropy). If the same fluorophore starts coupling energy to neighboring fluorophores tagged to cells that are randomly oriented, the latter fluorophores will start to fluoresce and emit light which has a lower degree of polarization (i.e. indicating low anisotropy). As time passes the degree of anisotropy will therefore decay.
Fluorescence lifetime imaging microscopy (FLIM) is an imaging technique based on differences in the average decay rate of excited states from a fluorescent sample. The contrast in a FLIM image is thus based on the lifetime of individual fluorophores rather than their emission spectra. Unlike intensity measurements, fluorescence lifetime measurements do not depend on: concentration, absorption by the sample, sample thickness, photo-bleaching, and/or excitation intensity.
The fluorescence lifetime of a fluorophore is the average decay rate of excited states from a fluorescent sample, and is characteristic for each fluorescent molecule. As a result, it can be used to characterize a sample. The fluorescent lifetime, however, is dependent on the local environment of the fluorophore, including: FRET, quenching, molecular rotation pH, ion or oxygen concentration, molecular binding or proximity of energy acceptors, as such it is possible to ascertain a wealth of information from the fluorophore by measuring its lifetime. FLIM is often used to observe a change, typically a reduction, in the fluorescence lifetime of a donor, when different fluorophores are in close proximity.
FRET is a process which describes the non-radiative transfer of energy between two similar energy systems that lie physically close together. For example, a donor fluorophore, which is initially in an excited state may transfer energy to an acceptor fluorophore through non-radiative dipole-dipole coupling. In doing so, the acceptor enters an excited state, with the donor becoming quenched. The efficiency of this energy transfer is extremely sensitive to small changes between the distance of the donor and acceptor, and is inversely proportional to the sixth power of that distance. This results in changes to the fluorescence intensity and the fluorescence lifetimes of the two fluorophores.
Two forms of FRET which are of importance are homo-FRET and hetero-FRET. In homo-FRET, only one type of fluorophore is present, as such the energy transfer is reversible. This results in the fluorescent emission form the fluorophore having largely the same polarization as that of the incident excitation beam. In hetero-FRET, two types of fluorophores are present (for example, A, B), as such the fluorophores become mixed into a combination of pairs, for example AA, AB, BA, and BB. If the excitation is tuned to the absorption peak of A, the fluorescence consists of contributions from A (homo-FRET), and B (hetero-FRET). The homo-FRET and hetero-FRET emission may be spectrally separated and thus the hetero-FRET signal is more depolarized than the homo-FRET signal. In hetero-FRET, the fluorescence lifetime of the donor changes as a function of distance between the donor and acceptor, typically the closer the acceptor is to the donor, the shorter the fluorescence lifetime of the donor.
Typically FAIM (both steady-state, and time-resolved) is used to measure homo-FRET, while FLIM is typically used to measure hetero-FRET.
Currently, the systems used in this field are both bulky and expensive. Some embodiments aim to address or at least mitigate this.